The present invention relates generally to synthetic analogues of the tridecapeptide hormone .alpha.-melanotropin and more particularly to analogues which exhibit increased potency, prolongation and serum stability characteristics in comparison to the native hormone and which retain biological activity when covalently bonded to other elements or compounds.
.alpha.-Melanotropin (.alpha.-MSH, .alpha.-melanocyte stimulating hormone) is a tridecapeptide synthesized and secreted by the pars intermedia of the vertebrate pituitary. By convention, the amino acid residues of this tridecapeptide are numbered sequentially from the amino terminal (here acetyl substituted) carbon atom through the carboxy terminal (here carboxamide terminal) as follows: EQU Ac-Ser.sup.1 -Tyr.sup.2 -Ser.sup.3 -Met.sup.4 -Glu.sup.5 -His.sup.6 -Phe.sup.7 -Arg.sup.8 -Trp.sup.9 -Gly.sup.10 -Lys.sup.11 -Pro.sup.12 -Val.sup.13 -NH.sub.2.
In this formula the following abbreviations are used: Ser=serine; Tyr=tyrosine; Met=methionine; Glu=glutamic acid; His=histidine; Phe=phenylalanine; Arg=arginine; Trp=tryptophan; Gly=glycine; Lys=lysine; Pro=proline; Val=valine.
.alpha.-MSH reversibly darkens amphibian skins by stimulating melanosome movement (dispersion) within melanophores. .alpha.-Melanotropin also affects both normal and transformed (melanoma) mammalian melanocytes by stimulating adenylate cyclase activity, tyrosinase activity and melanin production. In addition, recent studies suggest that the native hormone may have important functions in fetal development and in neural mechanisms related to learning and memory. See, Front. Horm. Res., 4, (Tilders, et al., Eds.), S. Karger, Basil (1977).
The recognition that .alpha.-MSH functions in a number of roles in mammals, including humans, in addition to its well-characterized role in the color change mechanism of poikilothermic vertebrates and its effect on melanoma cell activity and growth, has prompted substantial research into production and testing of synthetic .alpha.-MSH and analogues thereof.
Variation in biological effects of .alpha.-MSH analogues are generally discussed in terms of "potentiation" (increased activity relative to naturally-occurring .alpha.-MSH) and "prolongation". Such effects are measured by the classic frog skin bioassay system. See, Shizume, et al., Endocrinology, 54, 553-560 (1954). Because .alpha.-MSH has an extremely short half-life in serum (about 2 minutes), synthetic analogues having greater serum stability in addition to greater potency and prolongation characteristics have been sought. Finally, because the hormone is essentially inactivated upon attempted iodination for purposes of generating a radiolabelled compound synthetic analogues capable of retaining biological activity after such processing have also been sought.
Of particular interest to the background of the invention are early studies noting the effects of heat-alkali treatment of naturally-isolated and synthetic .alpha.-MSH. Briefly put, products of such treatment lost optical activity due to varying degrees of randomized racemization of amino acids from L- to D-isomeric configurations. The treated hormones, however, exhibited significantly prolonged and retarded biological activities. It has been reported, for example, that in vivo and in vitro treatment of frog skins with heat-alkali treated .alpha.-MSH, .beta.-MSH or ACTH resulted in prolonged melanotropic (skin darkening) activity. Maximum prolongation and potentiation for .alpha.-MSH resulted when the hormone was heated at 60.degree. C. for 40 minutes in a solution of 0.1 N NaOH, and it was implicitly assumed that the biological effect was due to racemization of one or more residues in the peptide. See, e.g., Geschwind, et al. Arch. Biochem. Biophys., 106, pp. 200-206 (1964); Lerner et al, Exerpta Medica Int. Congr. Series 83, pp. 392-397 (1964); and Lande, et al., Biochem. Biophys. Acta, 251, pp. 246-253 (1971). As long ago as 1967 it was noted that: "It is important that .alpha.-MSH be made with D-amino acids in one or two places. If the site of racemization can be pinpointed, it may be possible to produce well-characterized peptides more active than the natural hormone" [Lande, et al., Pharm. Rev., 19, pp. 1-20 (1967)].
Research consistent with such suggestions, however, has provided less than satisfactory results. As one example, the synthetic analogue of .alpha.-MSH containing L-configuration amino acids at all but position 2 (i.e., [D-Tyr.sup.2 ]-.alpha.-MSH) was found to be significantly less potent than the natural hormone. See, Sawyer, et al., "Peptides: Structure and Biological Function," Proc. 6th Am. Pept. Symp. (Gross, et al., eds.) pp. 1017-1020, Pierce Chem. Co., Rockford, Ill. (1979). Similarly, it has been reported that the synthetic analogue of .alpha.-MSH containing D-Ser.sup.1 was no more active than the native hormone. See, Eberle, et al., Helv. Chim. Acta, 62, pp. 2460-2483 (1979).
Also of interest to the background of the present invention is the finding that substitution of aliphatic aminoacids such as norleucine (Nle) for methionine at position 4 resulted in more potent biological activity. [Nle.sup.4 ]-.alpha.-MSH was found to be approximately twice as potent as .alpha.-MSH both in dispersing amphibian melanophores and in stimulating melanoma adenylate cyclase activity. See, Sawyer, et al., supra. The Nle.sup.4 analogue was found to be resistant to inactivation by chloramine-T, an oxidant used in peptide iodination, but was inactivated upon further contact with sodium iodide. See, Heward, et al., Hoppe-Seyler's Z. Physiol. Chem., 360, pp. 1851-1859 (1979).
There continues to exist, therefore, a longstanding need in the art for serum-stable, highly potent .alpha.-MSH analogues which have prolonged biological activity and which retain biological activity upon iodination processing.
Specifically incorporated by reference herein for the purpose of illustrating the background of the invention and the prior art is the publication by the inventors and their co-workers appearing in P.N.A.S. 77, pp. 5753-5758 (October 24, 1980).